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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb - 23 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his operon (S. typhimurium), trp operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: distilled water was used as solvent/vehicle, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene (10-50 µg/plate)
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
other: daunomycin (6 µg/plate); ICR 191 acridine (1 µg/plate)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
INITIAL TEST
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

CONFIRMATORY TEST
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48-72 h
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies on the test plates compared to the solvent/vehicle control plates
Evaluation criteria:
In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose response is a further criterion for mutagenic materials.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was seen at all test material concentrations, which did not influence the results of the assay

RANGE-FINDING/SCREENING STUDIES: A preliminary experiment was conducted to determine cytotoxicity and solubility of the test substance. Starting with the test material concentration of 5 mg/plate (recommended by OECD GL 471) and subsequent serial dilutions (ratio 1:2) following concentrations of test material were applied: 5, 2.5, 1.25, 0.625, 0.313 and 0.156 mg/plate on two bacteria strains (TA 98 and TA 1535) with different mutation type (frameshift and base-pair substitution) with and without metabolic activation.
The highest concentration of that solution was chosen for the main test that caused low cytotoxicity and/or where the precipitate did not interfere with the scoring according to OECD GL 471.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were acceptable and compatible with the historical control values.

Any other information on results incl. tables

Test Material concentrations:

Concentration 1: 5 mg/plate

Concentration 2: 2.5 mg/plate

Concentration 3: 1.25 mg/plate

Concentration 4: 0.625 mg/plate

Concentration 5: 0.313 mg/plate

 

 

Table 2:Mean plate counts and standard deviations (SD); strain: TA98

 

Controls/ test substance conc.

Colony count

TA98

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

18

2

13

2

NK+S9

23

1

20

2

PK

508

36

632

39

PK1+S9

2154

207

2032

116

PK2+S9

139

20

160

11

Conc. 1

18

4

14

2

Conc. 1+S9

21

2

23

4

Conc. 2

17

2

12

4

Conc. 2+S9

25

5

24

7

Conc. 3

20

3

16

8

Conc. 3+S9

30

4

16

3

Conc. 4

15

5

15

1

Conc. 4+S9

25

6

18

1

Conc. 5

22

4

12

3

Conc. 5+S9

27

6

23

3

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (daunomycin)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

 

Table 3:Mean plate counts and standard deviations (SD); strain: TA100

 

Controls/ test substance conc.

Colony count

TA100

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

82

1

87

16

NK+S9

100

10

96

4

PK

508

52

775

202

PK1+S9

1966

109

1783

111

PK2+S9

686

77

724

95

Conc. 1

87

8

84

7

Conc. 1+S9

100

8

85

12

Conc. 2

100

10

79

11

Conc. 2+S9

103

5

93

8

Conc. 3

82

6

80

6

Conc. 3+S9

103

7

88

9

Conc. 4

90

8

82

2

Conc. 4+S9

97

2

94

16

Conc. 5

97

4

79

3

Conc. 5+S9

109

17

89

10

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

PK2 - positive control 2 with S9 (benzo(a)pyrene)

Conc. - concentration

 

 

Table 4:Mean plate counts and standard deviations (SD); strain: TA1535

 

Controls/ test substance conc.

Colony count

TA1535

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

10

3

8

4

NK+S9

9

2

11

4

PK

375

33

312

28

PK1+S9

82

10

47

17

Conc. 1

8

2

8

3

Conc. 1+S9

9

2

9

7

Conc. 2

10

4

8

4

Conc. 2+S9

5

4

10

0

Conc. 3

11

8

14

3

Conc. 3+S9

5

1

8

3

Conc. 4

8

6

9

3

Conc. 4+S9

7

2

6

2

Conc. 5

10

3

11

3

Conc. 5+S9

8

2

9

1

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (sodium azide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

 

Table 5: Mean plate counts and standard deviations (SD); strain: TA1537

 

Controls/ test substance conc.

Colony count

TA1537

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

7

2

4

3

NK+S9

7

3

7

4

PK

1635

439

122

15

PK1+S9

144

23

177

29

Conc. 1

7

4

6

3

Conc. 1+S9

6

5

5

1

Conc. 2

7

2

5

2

Conc. 2+S9

6

0

6

1

Conc. 3

6

1

7

1

Conc. 3+S9

10

2

6

1

Conc. 4

8

1

6

4

Conc. 4+S9

8

3

5

1

Conc. 5

6

3

3

2

Conc. 5+S9

10

2

7

5

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (ICR 191 acridine)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

 

 

Table 6:Mean plate counts and standard deviations (SD); strain: WP2uvrA

 

Controls/ test substance conc.

Colony count

WP2uvrA

Main test:

Preincubation method

Confirmatory test:

Plate incorporation method

Mean

SD

Mean

SD

NK

17

2

27

4

NK+S9

13

2

31

4

PK

1000

30

1350

38

PK1+S9

402

31

357

44

Conc. 1

18

3

35

2

Conc. 1+S9

16

8

35

4

Conc. 2

17

5

35

3

Conc. 2+S9

16

4

29

2

Conc. 3

18

6

25

3

Conc. 3+S9

17

8

32

2

Conc. 4

16

1

26

5

Conc. 4+S9

16

1

36

11

Conc. 5

20

8

34

2

Conc. 5+S9

17

6

35

5

 

Key:

SD – Standard deviation

NK - negative control

PK - positive control without S9 (4-nitroquinoline-1-oxide)

PK1 - positive control 1 with S9 (2-aminoanthracene)

Conc. - concentration

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material did not induce reverse gene mutations in Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) or Escherichia coli (WP2 uvrA) tester strains. Therefore, the test material is considered to be not mutagenic in bacteria.